Ultrasmall superparamagnetic iron oxide nanoparticles and uses thereof

ABSTRACT

The present invention provides a biomimetic contrast agent comprising an amine-functionalized iron (II) oxide/iron(III) oxide nanoparticle core a targeting ligand attached to the nanoparticle core via a linker and an inert outer layer of a hydrophilic polymer conjugated to the targeting ligand and imaging methods using the biomimetic contrast agents. Also, provided is a dual functional contrast agent comprising a metal-doped iron (II) oxide/iron(III) oxide nanoparticle core, an inert layer of gold coating the nanoparticle core and a biodegradable cationic polymer linked thereto. The dual functional contrast agent is complexed to a therapeutic gene and when transfected into mesenchymal stem cells comprises a dual contrast agent and gene delivery system. In addition, methods of using the dual functional system are provided. Furthermore, kits comprising the biomimetic contrast agents and the dual contrast agent and gene delivery system are provided.

CROSS-REFERENCE TO RELATED APPLICATION

This non-provisional application claims benefit of provisional application U.S. Ser. No. 61/002,201 filed on Nov. 7, 2007, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the fields of magnetic resonance imaging and contrast agents and disease diagnosis and treatment. More specifically, the invention relates to iron oxide nanoparticles that are either doped or not doped with varying amounts of metal ions and, optionally, gold-coated, and are surface-modified with a biomimetic and bioresponsive entity that imparts specificity of the contrast agent, which may include delivery of a therapeutic gene, to the desired target.

2. Description of the Related Art

In recent years, magnetic nanoparticles (MNPs) have generated significant interest within the scientific community due to their huge range of potential applications such as media materials for storage systems (1-2), biotechnology (3-4), magnetic separation (5-6) targeted drug delivery (7-8) and vehicles for gene and drug delivery (9-12). Among the various MNPs, undoped and transition metal-doped, e.g., Magnetite or Maghemite, iron oxide nanoparticles, Fe₃O₄, g-Fe₂O₃ and M_(x)Fe_(y)O_(z) have found many applications in the area of biomedical diagnostics and therapy.

In the field of imaging, the superparamagnetic nature of iron oxide nanoparticles enables their use as potential contrast agents for magnetic resonance imaging (MRI). These nanoparticles with very large magnetic susceptibilities strongly influence the T₁ and T₂ relaxation of water molecules surrounding these MRI contrast agents. In addition, the magnetic properties of these iron oxide nanoparticles are also dependent on particle size. Iron oxide nanoparticles that are below 15-20 nm retain superparamagnetism and influence the T₂ relaxation to the largest extent (13-14). This influence on the T₂ relaxation highly modulates the MRI properties of the iron oxide nanoparticles. Hence, by adjusting the core size of the nanoparticles the magnetic properties for MRI can be enhanced. Typically, MNPs are synthesized with a particle size of 10-500 nm in diameter for biomedical applications.

In addition to size, the biocompatibility, solubility, and monodispersity of these MNPs are also critical for their use in vivo (15). However, the surface-chemical properties of MNPs do not facilitate the conjugation of biomolecules. Therefore, the surface of these MNPs must undergo modification or functionalization to enable the chemistry needed for coupling biomolecules to MNPs. In other words, in order to make the magnetic nanoparticles efficient delivery vehicles, introduction of suitable functional groups onto the surface of the particle is essential so as to facilitate the conjugation of molecules that will increase its solubility and increase its availability for various conjugation processes. Also, the conjugation process has to be efficient, yielding a stable product, which does not compromise the activity of the biomolecules. Moreover, the signal of the nanoparticles for imaging or other analytical techniques should not be affected as a result of the conjugation. In addition, these nanoparticles should also have maximum surface area so as to facilitate the binding of a maximum number of linkage sites on the surface.

In the United States, breast cancer is second to lung cancer as the leading cause of cancer death in women. This year alone, ˜40,000 women will die from the disease despite a decline in the death rates. Detection is the best method to prevent mortality but only 60% of cancers are detected at the earliest, subclinical stages of the disease. Also, ovarian cancer has the highest mortality rate of all gynecological malignancies. Poor prognosis of the disease is directly related to late detection after peritoneal tumor dissemination and the formation of ascites. Failure to detect early primary tumors or metastases results in very poor clinical outcome.

MRI is a powerful imaging modality for detection of cancer and other diseases because it provides high spatial resolution and excellent soft tissue contrast. However, MRI imaging systems need to be developed that are sensitive enough to detect cancers in the early stage of development and to detect metastatic cancers.

Thus, there is a recognized need in the art for improved contrast agents effective to detect early stage primary and metastatic cancers via magnetic resonance imaging, including contrast agents suitable to deliver a therapeutic gene during imaging. More specifically, the prior art is deficient in biomimetic contrast agents and a dual functioning contrast agent/gene/drug delivery system effective to specifically target primary and metastatic cancer cells with MRI. The present invention fulfills this long-standing need and desire in the art.

SUMMARY OF THE INVENTION

The present invention is directed to a biomimetic contrast agent. The contrast agent comprises an amine-functionalized iron (II) oxide/iron(III) oxide nanoparticle core, a targeting ligand attached to the nanoparticle core via a linker and an inert outer layer of a hydrophilic polymer conjugated to the targeting ligand. The present invention is directed to a related biometric contrast agent further comprising a metal doping agent in the nanoparticle core. The present invention is directed to another related biometric contrast agent further comprising a gold coating on the nanoparticle core.

The present invention also is directed to a dual contrast agent that comprises a metal-doped iron (II) oxide/iron(III) oxide nanoparticle core, an inert layer of gold coating the nanoparticle core and a biodegradable cationic polymer linked thereto. The present invention is directed to a related contrast agent further comprising a DNA encoding an anti-tumor cytokine complexed with the cationic polymer.

The present invention is directed further still to a kit comprising the biomimetic contrast agent described herein. The present invention is directed further still to a kit comprising the dual contrast agent described herein or the dual contrast agent complexed to a DNA encoding a therapeutic gene described herein. The present invention is directed to a related kit further comprising the buffers and reagents effective to transfect mesenchymal stem cells with the contrast agent-DNA complex.

The present invention is directed further still to a dual contrast agent and gene delivery system. The system comprises mesenchymal stem cells (MSCs) transfected with the dual contrast agent of described herein complexed with a DNA encoding an anti-tumor cytokine.

The present invention is directed further still to an in vivo method using magnetic resonance imaging for detecting an early stage primary or metastatic cancer in a subject. The method comprises administering to the subject a sufficient amount of the biomimetic contrast agent described herein to provide a detectable contrast image of the contrast agent within the primary or metastatic cancer therein, wherein a location of the image correlates to a location of the cancer.

The present invention is directed further still to a method for reducing metastasis of tumor cells in a subject. The method comprises administering to the subject an amount of the mesenchymal stem cells (MSCs) comprising the dual contrast agent and gene delivery system described herein sufficient to target metastatic tumor cells and simultaneously delivering the contrast agent and the anti-tumor cytokine thereto. A magnetic resonance image of the contrast agent within the metastatic tumor cells is obtained; where simultaneously imaging the contrast agent and delivering the anti-tumor cytokine locates a site of metastatic tumor cells and induces a pro-inflammatory response against the same, thereby reducing metastasis of the tumor cells. The present invention is directed to a related method further comprising implementing one or both of a surgical regimen or one or more other chemotherapeutic regimens.

Other and further aspects, features and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages and objects of the invention, as well as others which will become clear, are attained and can be understood in detail, more particular descriptions of the invention are briefly summarized. The above may be better understood by reference to certain embodiments thereof which are illustrated in the appended drawings. These drawings form a part of the specification. It is to be noted; however, that the appended drawings illustrate preferred embodiments of the invention and therefore are not to be considered limiting in their scope.

FIGS. 1A-1B depict the syntheses of Gadolinium-doped Ultrasmall Paramagnetic Iron Oxide Nanoparticles (GdUSPIO) (FIG. 1B) and amine-functionalized Gadolinium-doped Ultrasmall Paramagnetic Iron Oxide Nanoparticles (amine-GdUSPIO) (FIG. 1B).

FIGS. 2A-2C are the FTIR spectrum of USPIO and amine-USPIO (FIG. 2A) and the TEM image of USPIO (FIG. 2B) and amine-USPIO (FIG. 2C).

FIG. 3 shows results for the ninhydrin assay for the quantitative determination of the conjugation of 3-aminopropyltrimethoxysilane (APTMS) to ultra small paramagnetic iron oxide nanoparticles

FIGS. 4A-4C depict the synthesis of gold coated doped iron oxide nanoparticles (FIG. 4A) and the electronic absorption spectra of various gold coated doped iron oxide nanoparticles (FIG. 4B) and TEM images of gold-coated doped nanoparticles (FIG. 4C).

FIG. 5A-5D depict the Fourier Transform-infrared (FT-IR) vibrational spectra of synthesized amine-GdUSPIO (top) and GdUSPIO (bottom) nanoparticles (FIG. 5A), the X-Ray Diffraction (XRD) pattern of synthesized GdUSPIO (top) and amine-GdUSPIO (bottom) nanoparticles (FIG. 5B) and the Transmission Electron Microscopy (TEM) images of synthesized (FIG. 5C) GdUSPIO and (FIG. 5D) amine-GdUSPIO nanoparticles.

FIGS. 6A-6E depict the ¹H NMR spectra of homobifunctional COOH-PEG400-COOH diacid (b), the HPLC spectra for separation of bioresponsive peptide with the RGDS (GRGDSGPQGLAG) ligand on an analytical column (b) and a preparative column showing the main peaks of interest at 25 and 19 min (FIG. 6C) along with smaller peaks representing byproducts of the synthesis reaction, the ¹H NMR spectrum of synthesized COOH-PEG₂₀₀₀-OMe outer stealth layer showing proton peaks of the polymer (FIG. 6D), the relaxivities for ultra small paramagnetic iron oxide nanoparticles (r=130.8 mMol/lsec), amine-functionalized ultra small paramagnetic iron oxide nanoparticles (r=5.5 mMol/lsec) and internal control CuSO₄ (r=1.2 mMol/lsec) (FIG. 6E).

FIGS. 7A-7E depict synthetic schema for amine-functionalized iron oxide nanoparticles (FIG. 7A) HOOC-TEG-t-butyl acrylate synthesis (FIG. 7B), peptide synthesis (FIG. 7C), USPIO-TEG-COOH synthesis (FIG. 7D), and USPIO-TEG-Peptide-mPEG₅₀₀₀ synthesis (FIG. 7E).

FIGS. 8A-8F depict the NMR spectra of OH-TEG-Ots (FIG. 8A), OH-TEG-N₃ (FIG. 8B), t-butyl ester-TEG-N₃ (FIG. 8C), NH₂-TEG-t-butylester (FIG. 8D), t-butylester-TEG-COOH (FIG. 8E), and the MALDI-TOF data for dual bioresponsive peptide (FIG. 8F).

FIG. 9 is a schematic representation of IL-12 protein from nanoparticle-transfected MSCs homing to tumor cells.

FIGS. 10A-10B depict the TEM image of synthesized Au-coated 4% Gd[III]-doped iron oxide nanoparticles where scale=20 nm (FIG. 10A) and the MRI images of new contrast agents; Samples 1-7=CUSO₄; 8-9=4%, 10-11=0%, and 12-13=2% iron oxide-doped Gd nanoparticles (FIG. 10B).

FIG. 11 depicts the ¹H NMR spectrum of synthesized reducible LLC-N-boc conjugated polymers.

FIG. 12 depicts the gel retardation assay of deprotected reducible LLC copolymers: 1=free DNA; 2=linear PEI; 3=branched PEI; 4=PLL; 5=1/1; 6=5/1; 7=10/1; 8=15/1; 9=20/1; 10=25/1; 11=30/1; 12=40/1; 13=50/1; and 14=100/1 N/P ratios.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one.

As used herein “another” or “other” may mean at least a second or more of the same or different claim element or components thereof. Some embodiments of the invention may consist of or consist essentially of one or more elements, method steps, and/or methods of the invention. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.

As used herein, the term “subject” refers to any recipient of the contrast agents provided herein.

In one embodiment of the present invention there is provided a biomimetic contrast agent, comprising an amine-functionalized iron (II) oxide/iron(III) oxide nanoparticle core; a targeting ligand attached to the nanoparticle core via a linker; an inert outer layer of a hydrophilic polymer conjugated to the targeting ligand. Further to this embodiment the biomimetic contrast agent comprises a metal doping agent in the nanoparticle core. Examples of a metal doping agent are gadolinium, manganese or cobalt. In another further embodiment the biomimetic contrast agent comprises a gold coating on the nanoparticle core.

In all embodiments the ratio of the iron (II) to the iron(III) in the nanoparticle core may be about 1:1. Also, in all embodiments the linker may comprise a triethylene glycol polymer or a polyethylene glycol polymer. In addition, the inert outer layer may comprise a polyethylene glycol polymer.

Also, in all embodiments the targeting ligand may comprise a contiguous peptide sequence of an enzyme cleavable sequence and a targeting sequence in tandem. Further to these embodiments the targeting ligand may comprise a blocking agent bound to a terminal sidechain amino acid of the peptides. In an aspect of these embodiments the enzyme cleavable sequence may be cleavable by an enzyme associated with ovarian cancer cells. For example, the enzyme cleavable sequence may be a metalloproteinase-13 cleavable sequence, particularly the sequence shown in SEQ ID NO: 1. In another aspect the targeting sequence may be an endothelin-1 (ET-1) sequence, particularly the sequence shown in SEQ ID NO: 2, an integrin-binding sequence shown in SEQ ID NO: 3, or a human immunodeficiency virus Tat peptide sequence shown in SEQ ID NO: 4. Also in this aspect, the targeting ligand may have the contiguous sequences shown in one of SEQ ID NOS: 5-7.

In a related embodiment the present invention provides a kit comprising the biomimetic contrast agent as described supra.

In another embodiment the present invention provides an in vivo method using magnetic resonance imaging for detecting an early stage primary or metastatic cancer in a subject, comprising administering to the subject a sufficient amount of the biomimetic contrast agent described supra to provide a detectable contrast image of the contrast agent within the primary or metastatic cancer therein, wherein a location of the image correlates to a location of the cancer. Representative cancers may be ovarian cancer or breast cancer or other cancer expressing MMP-13 enzyme.

In yet another embodiment the present invention provides a contrast agent, comprising a metal-doped iron (II) oxide/iron(III) oxide nanoparticle core; an inert layer of gold coating the nanoparticle core; and a biodegradable cationic polymer linked thereto. Further to this embodiment the contrast agent comprises DNA encoding an anti-tumor cytokine complexed with the cationic polymer. In both embodiments the anti-tumor cytokine may be interleukin-12. Also, the doping metal may be gadolinium (III), manganese, cobalt, or ruthenium. In addition, the cationic polymer may comprise disulfide-reducible, linear, L-lysine-modified copolymers (LLC).

In a related embodiment the present invention provides a kit comprising the dual contrast agent described supra or this dual contrast complexed to a DNA encoding an anti-tumor cytokine. Further to this related embodiment the kit comprises the buffers and reagents effective to transfect mesenchymal stem cells with the contrast agent-DNA complex.

In another related embodiment the present invention provides a dual contrast agent and gene delivery system, comprising mesenchymal stem cells (MSCS) transfected with the contrast agent described supra complexed with a DNA encoding an anti-tumor cytokine. An example of an anti-tumor cytokine is interleukin-12.

In yet another embodiment of the present invention there is provided method for reducing metastasis of tumor cells in a subject, comprising administering to the subject an amount of the mesenchymal stem cells (MSCs) comprising the dual contrast agent and gene delivery system described herein sufficient to target metastatic tumor cells; simultaneously delivering the contrast agent and the anti-tumor cytokine thereto; and obtaining a magnetic resonance image of the contrast agent within the metastatic tumor cells; wherein simultaneously imaging the contrast agent and delivering the anti-tumor cytokine locates a site of metastatic tumor cells and induces a pro-inflammatory response against the same, thereby reducing metastasis of the tumor cells. Further to this embodiment the method comprises implementing one or both of a surgical regimen or one or more other chemotherapeutic regimens.

The present invention provides biomimetic ultrasmall paramagnetic iron oxide (USPIO) nanoparticles (USPIO) with dual bioresponsive elements effective to selectively target tumor cells in the preclinical stage of development for early detection of cancer for use in magnetic resonance imaging. Such contrast agents act as early detection probes with broad availability to patients and lead to life saving early detection and treatment of cancer. The USPIO nanoparticles enhance sensitivity during magnetic resonance imaging (MRI) because of increased specificity for the target by providing a modifiable surface. The presence of two levels of targeting, based on specific tumor biochemistry, significantly increases the targeting of the probe to the tumor that would overcome the hurdles of late detection, safety, rapid elimination, and non-specific extravasation.

The contrast agent comprises an amine-functionalized nanoparticle core of an undoped or metal-doped iron oxide, a linker, a targeting ligand and an outer inert or stealth layer. The nanoparticle core may be an Fe(II)/Fe(III) oxide, such as which is biodegradable. The nanoparticles may be about 10-100 nm, where ultrasmall nanoparticles are less than 50 nm, preferably about 20 nm to about 50 nm in diameter. For example, Fe(II)/Fe(III) oxide may be present in the core in a 1:1 ratio. A suitable doping metal is, but not limited to, gadolinium, manganese, cobalt, or ruthenium. The linker may comprise a short polyethylene glycol polymer (PEG), for example, a PEG₄₀₀ polymer, or a triethylene glycol polymer. Optionally, the nanoparticle core may be coated with an inert layer of gold.

The targeting ligand is covalently attached, conjugated or linked to the nanoparticle core via the linker. The targeting ligand comprises two contiguous peptide targeting sequences. The first targeting peptide sequence is cleavable by an enzyme associated with a cancer. For example, the cancer may be any cancer expressing the metalloproteinase-13 enzyme, such as ovarian cancer, and the peptide may be cleavable by metalloproteinase-13 enzyme. This peptide sequence may comprise a PQGLA sequence (SEQ ID NO: 1). The second targeting peptide sequence is a cancer cell specific peptide that binds to the tumor cells. For example, the second targeting peptide sequence may be an endothelin-1 (ET-1) sequence, such as CSCSSLMDKECVYFCHLDIIW (SEQ ID NO: 2) or an integrin-binding sequence, such as an RGDS peptide sequence (SEQ ID NO: 3) or a human immunodeficiency virus (HIV) Tat peptide sequence, such as YGRKKRRQRRR (SEQ ID NO: 4). The peptides comprising the targeting sequences may be blocked, that is, the amino acid side chains comprising the peptides may be blocked by a blocking agent selective for the amino acid. The peptides are synthesized as described in Example 3.

Thus, a contiguous targeting ligand sequence may comprise the MMP-13 cleavable sequence PQGLA and the ET-1 sequence as GCSCSSLMDKECVYFCHLDIIWGPQGLAG (SEQ ID NO: 5), the integrin-binding sequence as GRGDSGPQGLAG (SEQ ID NO: 6), and the HIV Tat sequence as GYGRKKRRQRRRGPQGLAG (SEQ ID NO: 7). Glycine residues are added as spacers at the beginning and end of the contiguous sequence and between the first and second targeting sequences to increase enzyme recognition of the sequence. The nanoparticle-targeting ligand conjugate further comprises an inert or stealth outer layer, such as a hydrophilic polymer, for example, but not limited to, a polyethylene glycol polymer conjugated to the targeting ligand. The PEG polymers may be a PEG₂₀₀₀ to about PEG₅₀₀₀.

As a representative example, the contrast agents disclosed herein take advantage of the presence and role of metalloproteinase-13 (MMP-13) in ovarian cancer and other cancers, such as breast cancer, because this enzyme is active within these carcinomas that also express specific cellular receptors. The endothelin receptor (ET_(A)R) and endothelin-1 (ET-1) peptide are produced in both primary and metastatic ovarian tissue as well as the integrin (α_(v)β₃) receptors that bind the arginine-glycine-aspartic acid-serine (RGDS) sequence. In addition, the HIV Tat protein transduction domain has been shown to be a very effective and promiscuous cell penetrating peptide.

In general, upon administering the biomimetic contrast agent to a subject, the outer layer prevents non-specific cellular uptake of the contrast agent as well as to limit premature interactions with blood and immune components. At the first level of targeting, the enzyme cleavable peptide sequence is recognized and cleaved only within the tumor compartment by the enzyme which releases the outer protective stealth layer. Release of the stealth layer reveals, as the second level of targeting, the second peptide sequence, which as a ligand, specifically binds to the tumor cells for cellular uptake of the contrast agents for magnetic resonance imaging.

The present invention also provides a dual contrast agent and gene delivery system useful with a MRI imaging modality. The system comprises a nanoparticle-based platform (USPIO) to enable detection and cell-based gene therapy treatment of metastatic cancer, such as, but not limited to, metastatic breast cancer. The contrast agent may comprise the gold-coated metal-doped iron oxide nanoparticle core described herein. Particularly, a gadolinium-doped, such as gadolinium (III), iron oxide nanoparticle core is coated with an inert layer of gold.

The gold-coated nanoparticles are conjugated to cationic polymers that may comprise novel disulfide-reducible, linear, L-lysine-modified copolymers (LLC). The cationic polymers are effective to form a complex with a therapeutic gene, particularly a chemotherapeutic gene. Examples of therapeutic genes may be interleukins, such as, interleukin-12. It is contemplated that plasmid DNA encoding the therapeutic gene is complexed with the cationic polymer using well-known and standard molecular biological techniques.

The cationic polymer-AuGdUSPIO nanoparticle conjugate further functions in a gene or drug delivery system. These cationic nanoparticles can transfect mesenchymal stem cells (MSCs) using standard molecular biological techniques. MSCs demonstrate homing properties to tumor cells, have been used as gene delivery vehicles, have reduced immunogenicity and are easy to expand and maintain in cell culture.

In addition, the present invention also provides kits comprising the contrast agents, including the contrast agent complexed to a DNA encoding a therapeutic DNA. Also, a kit comprising the contrast agent useful in the gene delivery system may further comprise those buffers, reagents, etc. standard in the art that are useful during transfection of a mesenchymal stem cell with the DNA-contrast agent complex.

As such, the present invention provides in vivo magnetic resonance imaging methods using the contrast agents described herein. These contrast agents may be administered in amounts sufficient to produce a detectable magnetic resonance image in cancerous cells or tissues or tumors. These methods are well suited to detect early stage primary or metastatic cancers, such as, but not limited to, ovarian or breast cancers, upon targeting thereto. One of ordinary skill in the art is well-suited to determine amounts of the contrast agents to administer to a subject, the route of administration and the MRI imaging conditions necessary to obtain a useable detectable image. Furthermore, such early detection greatly enhances the prognosis of the subject.

Also, the present invention provides methods of simultaneously detecting one or more sites of metastatic cancer cells and reducing metastasis of metastatic cells distal to a primary cancer or tumor using the dual contrast agent and gene delivery system described herein during MRI. The method is particularly useful in reducing metastasis of a breast cancer to or metastatic breast cancer cells in the bones, lung, liver, and brain of the subject. It is contemplated that delivery of the anti-tumor gene, such as, interleukin-12 would induce a pro-inflammatory response against the metastatic cancer cells. One of ordinary skill in the art is well able to determine the dosage of the transfected MSCs based on the type of metastatic cancer, the progression of the cancer, the health of the subject, etc. and the conditions for MRI are easily determined. Upon location of the metastatic cells in the subject, the methods further provide for additional therapeutic intervention, such as, surgical removal of cancerous tissue and/or one or more additional chemotherapeutic agent or drug regimens as are well-known and standard.

The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion.

EXAMPLE 1 Synthesis of Metal- and Undoped Ultrasmall Paramagnetic Iron Oxide Nanoparticles USPION

The synthesis of undoped iron oxide nanoparticles and gadolinium, manganese, cobalt, and ruthenium doped iron oxide nanoparticles were carried out using a modified procedure by Hong et al. (17) For metal-doped particles, the concentration of the gadolinium ions in the final product was 0, 2, 4, 6, 8, and 10% and was 8% for both manganese and cobalt to the total amount of the Fe³⁺/Fe²⁺ ions. Calculations were done such that the concentration of Fe²⁺/Fe³⁺=1. Calculated amount of FeCl₂.4H₂O (M.W: 198.81) and FeCl₃.6H₂O (M.W:270.3) and, for metal-doped nanoparticles, a stoichiometric amount of metal chloride salts were weighed and dissolved in ultra-pure water. Table 1 shows the amounts of FeCl₂.4H₂O, FeCl₃.6H₂O and gadolinium used in the nanoparticle core.

TABLE 1 Amount of Amount of FeCl₂•4H₂O FeCl₃•6H₂O 1.5 g Fe₃O₄ + 0 g Gd 0.6348 g 0.8651 g (0%) (3.2 mmol) (3.2 mmol) 1.47 g Fe₃O₄ + 0.03 g Gd 0.6221 g 0.8478 g (2%) (3.136 mmol) (3.136 mmol) 1.44 g Fe₃O₄ + 0.06 g Gd 0.6094 g 0.8305 g (4%) (3.072 mmol) (3.072 mmol) 1.41 g Fe₃O₄ + 0.09 g Gd 0.596 g 0.813 g (6%) (3.008 mmol) (3.008 mmol) 1.38 g Fe₃O₄ + 0.12 g Gd 0.5841 g 0.7959 g (8%) (2.944 mmol) (2.944 mmol) 1.35 g Fe₃O₄ + 0.15 g Gd 0.5713 g 0.778 g (10%) (2.88 mmol) (2.88 mmol)

After the complete dissolution of the reactants, the mixture was transferred to a three neck round bottom flask in an inert atmosphere. The reaction mixture was then heated to 60° C. for about 15-20 mins followed by the addition of liquid ammonia to a rapidly stirring mixture. Additional ammonia was added to adjust the pH of the solution in the range of 8-9, and the nanoparticles were allowed to grow for additional 90 minutes. After 90 minutes the reaction mixture was split in two 500 ml centrifuge tube and the nanoparticles obtained were centrifuged for 20 mins at 5000 r.p.m. Supernatant liquid obtained was discarded and nanoparticles obtained were washed 3 times with ultra pure water until it showed a negative test for the presence of chloride ions. After no more chloride ions were present, half of the particles were transferred to a three neck round bottom flask with Ethanol/Water as the suspending solvent for functionalization, while other half was stored in water in the 500 ml centrifuge tube for further characterization.

Synthesis of Amine-Functionalized Gadolinium-Doped Ultrasmall Paramagnetic Iron Oxide (Amine-GdUSPIO) Nanoparticles

FIGS. 1A-1B are synthetic schema for Gd-doped USPIOs and amine-GdUSPIOs. The metal-doped and undoped USPIO nanoparticles obtained in the previous step were transferred to a three neck round bottom flask and heated to 60° C. for about 15 minutes. After 15 minutes, 15 mls of 3-aminopropyltrimethoxysilane (APTMS) was added drop wise through addition funnel or with a syringe to the rapidly stirring mixture. The reaction mixture was further heated at 60° C. for about an hour with constant stirring to allow complete functionalization. After the completion of reaction, the reaction mixture was again transferred to 500 ml centrifuge tube and the mixture was centrifuged for about 20 minutes at 5000 r.p.m. The supernatant liquid was discarded and functionalized nanoparticles were washed three times with ethanol followed by washing with water, and finally the reaction mixture was stored in water for further characterization. FIG. 2A shows the IR spectrum of USPIO and amine-USPIO. Whereas the particle size measure by TEM was observed to be around 8-10 nm (FIGS. 2B-2C). Results for the quantitative determination of the conjugation of APTMS to ultra small paramagnetic iron oxide nanoparticle using a standard ninhydrin assay are shown in FIG. 3 and the values obtained with this assay are shown in Table 2.

TABLE 2 Samples Absrobance Blank Final Conc. Amine nmole/ml 0.173 0.066 0.107 46.52173913 0.142 0.066 0.076 33.04347826 0.124 0.066 0.058 25.2173913 0.091 0.066 0.025 10.86956522 Average 0.1325 0.066 0.0665 28.91304348 STDEV 0.034278273 0 0.03427827 14.90359696

Synthesize Au-coated M_(x)Fe₂O₃

FIG. 4A is the synthetic scheme for gold-coated doped USPIOs. 100 ml of metal (Gd, CO, or Mn) doped iron oxide nanoparticle was diluted to 1 ml with water and the particles were suspended with 2 mls of 0.1 M citric acid. Once the particles were suspended it was followed by the successive alternate addition of 1 ml of 1% HAuCl₄ and 2 ml of NH₂OH.HCl. The addition of 1% HAuCl₄ was increased to 2 ml after 1^(st) two additions. The amount of 0.1M NH₂OH.HCl was maintained same for all the five additions. After the solution turned faint purple or pink. Two more iterations of HAuCl₄ were added to precipitate all the particles which were then separated using magnet.

UV visible absorption spectra in FIG. 4B shows ˜530-575 nm absorption of energy by the gold coated nanoparticles in various samples. The presence of Gd, Mn and Co in various samples was characterized using ICP-AES. Table 3 shows the relative concentration of all the elements in various samples. Further, the concentration of Gd in most of the sample was displayed as 0 due to very low solubility of these gold coated samples into the solution which resulted in overall decrease in the detection limit of gadolinium. These particles were characterized with the particle size of 15-20 nm as shown in the TEM images in FIG. 4C.

TABLE 3 Samples Fe Gd Mn Co Au USPIO 118.20 0.00  4% Gd USPIO 114.40 0.38  6% Gd USPIO 109.54 0.41  8% Gd USPIO 101.50 9.20 10% Gd USPIO 87.50 10.53 Gold Coated Samples  2% Gd USPIO 15.4 0.08 0 0 65.93  4% Gd USPIO 2.15 0 0 0 36.33  6% Gd USPIO 1.5 0 0 0 63.25  8% Gd USPIO 2.9 0 0 0 53.58 10% Gd USPIO 2.4 0 0 0 28.3  8% Mn USPIO 40.7 0 1.3 0 3.09  8% Co USPIO 36.06 0 0 1.12 3.97

EXAMPLE 2 Spectroscopic Characterization of GdUSPIO and Amine-GdUSPIO Nanoparticles Fourier Transform-Infra Red (IR) Vibrational Spectrum

To confirm the presence of the amine groups present on amine-GdUSPIO, FT-IR spectroscopy was used for both GdUSPIO and amine-GdUSPIO. The FT-IR spectra were measured at room temperature with a Nicolet Avator 360 FT-IR spectrophotometer (Varian, Palo Alto, Calif.). Sample concentrations in the pellets were ˜1 mg of sample/200 mg of KBr salt. The synthesized MNPs were pressed into KBr pellets to record the solid state infrared spectra. To improve the signal-to-noise ratio, multiple scans (264) of each sample were collected and the slowly sloping baselines were subtracted from the digitally collected spectra using GRAMS/32 software package (Thermo Galactic, Inc. Salem, N.H.).

The combined FT-IR spectra of synthesized amine-GdUSPIO (FIG. 5A-top) and GdUSPIO (FIG. 5A-bottom) showed the spinel structure of GdUSPIO and amine-GdUSPIO with vibrational bands at 577 and 680 cm⁻¹, which corresponded to the stretching vibrations of magnetite (n(Fe—O)) having different symmetry. In addition, the vibrational band at 577 cm⁻¹ is attributed to the T_(2g) modes of vibrations and the vibrational band at 686 cm⁻¹ is attributed to the presence of A_(1g) modes of vibrations of the Fe—O. These vibrations in the IR region confirmed the presence of the magnetite crystal lattice.

The surface functionalization of the magnetite lattice was confirmed by the presence of the 2922 and 2957 cm⁻¹ vibrational bands in the IR spectrum of amine-GdUSPIO. These vibrational bands of amine-GdUSPIO can be attributed to the symmetric (n_(s)(CH)), and asymmetric stretching vibrations (n_(as)(CH)), respectively, which corresponds to the —CH₂ present in the APTMS molecule. The absorption band at 2843 cm⁻¹ can be attributed to the symmetric stretching vibrations of (n_(s)(O—CH₃)) of APTMS. The amine functionalized GdUSPIO was confirmed by the presence of the amine symmetric ((n_(s)(NH)) and asymmetric (n_(as)(NH)) stretching vibrations of —NH₂ at 3423 and 3393 cm⁻¹, respectively.

Finally, the presence of the symmetric stretching vibrations of the silane groups (n_(s)(Si—O)) at 991 cm⁻¹ in amine-GdUSPIO confirmed the presence of the silane molecules bonded to the metal oxide surface. This further confirmed the amine-functionalization of the GdUSPIO nanoparticles. The observed FT-IR vibrational frequencies for both GdUSPIO and amine-GdUSPIO are also tabulated in Table 4 for better comparison.

TABLE 4 Assigments GdUSPIO (cm⁻¹) amine-GdUSPIO (cm⁻¹) n(Fe—O) 577 577 n(Fe—O) 680 686 n_(s)(SiO) — 991 n_(s)(O—CH₃) — 2843 n_(as)(CH) — 2922 n_(s)(CH) — 2957 n_(s)(NH) — 3423 n_(as)(NH) — 3393

X-Ray Diffraction (XRD) Pattern

The phase morphology of the MNPs was identified from the XRD pattern of the nanoparticles that was obtained from the Siemen D5000 q/2q Diffractometer (Siemen, New York, N.Y.) using Cu Ka X-Ray line. Briefly, 1.0 mg of sample was suspended in isopropanol to form a concentrated slurry, which was then finely ground with a mortar and pestle. The sample was spread on to the sample holder to form a thin layer of nanoparticles and the XRD pattern was recorded by varying 2q from 15-77°. The tube voltage was kept at 40 kV and the current used was 30 mA at all time points. All the spectral figures were prepared with the IGOR Pro (version 6.0) software. The reflection at 311 lattice plane was used to estimate the average crystalline size by applying the Scherrer model. The Scherrer model is expressed as Equation 1:

d=0.9l/b cos q  Eq. (1)

where l=0.154 nm for Cu K_(a) line; d is the crystalline diameter, b is the full width at the half maximum of the 311 peak in radians, and q is the peak position in radians.

The spinel structure of both GdUSPIO and amine-GdUSPIO magnetite nanoparticles was confirmed from their XRD pattern. The XRD pattern of both GdUSPIO and amine-GdUSPIO is shown in FIG. 5B. The XRD pattern for both GdUSPIO and amine-GdUSPIO showed similar peaks for the pure phase of magnetite. In addition, the particles do not display sharp diffraction peaks, which are typically observed for amorphous and ultrafine materials. An average crystalline size of 8.45 nm was calculated using Eq. 1 for both GdUSPIO and amine-GdUSPIO nanoparticles.

Transmission Electron Microscope (TEM)

The particle size and the morphology of both GdUSPIO and amine-GdUSPIO were characterized using TEM. A carbon coated TEM grid (Pelco® No. 160) was used for nanoparticle visualization via a transmission electron microscope (Jeol Transmission Electron Microscopy, JEM-2010, Tokyo, Japan). Briefly, 20 μl of the nanoparticles suspended in water was pipetted on to a carbon-coated grid. The sample was allowed to settle on the grid for approximately 1 minute in a 100% humidified atmosphere. The carbon-coated grid was then washed with one drop of water, followed by the addition of another 20 μl of the nanoparticles onto the carbon coated grid. The grids were then left for approximately 45 seconds before gently removing excess sample through drying with a filter paper.

The TEM images of the GdUSPIO and amine-USPIO showed the presence of MNPs having a distinct spheroidal morphology and size. The particle size observed for both GdUSPIO and amine-GdUSPIO was found to be approximately 10-15 nm as shown in FIGS. 5C-5D, respectively. This suggests that modification of the nanoparticles does not affect the overall size and morphology of the nanoparticles. The TEM images also showed the agglomeration of the particles due to the particles being highly magnetic.

Inductively Coupled Plasma-Atomic Emission Spectroscopy: (ICP-AES)

The concentration of iron and gadolinium in all samples were measured with an inductively coupled plasma-atomic emission spectrophotometer (ICP-AES) instrument (Thermo, Waltham, Mass.). Briefly, 1.0 mg of the GdUSPIO and amine-GdUSPIO was digested in concentrated HCl for about 45 mins to dissolve. Once the particles were completely dissolved, the HCl was evaporated on a hot plate at 110° C. for 30 mins. The solid nanoparticles were then redissolved by heating with 1.0 mL of concentrated HNO3. Once the sample was dissolved, the solution was diluted 10 times with water to a final concentration of approximately 100 ppm. These solutions were finally used to obtain the ICP-AES data.

Elemental analysis by ICP-AES confirmed the presence of Gd ions in the nanoparticles. The ICP-AES data with respective concentrations in ppm and in moles for Fe, Gd, and Si are tabulated in Table 5. The elemental analysis by ICP-AES also showed the relative Si in the samples. The amount of the Gd in both 4 and 6% doped nanoparticles was found to be approximately 0.4 ppm as compared to 114 and 109.54 ppm of iron, respectively. This suggested that the effective doping of the Gd in 4 and 6% GdUSPIO is approximately 0.1%. Similarly, the amount of the doping in 8 and 10% Gd-doped USPIO was found to be 3.2 and 4.2%, respectively. This also corresponds to the doping efficiency of 42% in 10% GdUSPIO samples.

These discrepancies in the results can be attributed to the lower detection limit of the instrument for the amount of Gd present in the sample. The ICP-AES instrument can measure the Fe concentration in the range of 10-120 ppm, Hence, the samples were prepared considering the maximum concentration of 120 ppm for Fe²⁺/Fe³⁺ ions. However, this decreased the detection limit for the Gd ions as they were doped starting with the initial ratio of 4 and 6% and the actual doping was lower. Additionally, the molar ratio of the Gd in the sample decreased, as signal-to-noise ratio for the detection of Gd ions in the sample decreased. This led to an inaccuracy in the detection of the Gd at the lower limit. Furthermore, the molar ratio of the amount of the Fe to the amount of the Gd for 10% GdUSPIO was calculated to be 1.56:0.066 as compared to 2.98:0.02 shown before (16). The presence of the Si metal in amine-functionalized samples further confirmed the amine-functionalization of Gd-doped USPIO nanoparticles.

TABLE 5 Concentration in ppm Moles Sample Fe Si Gd Fe Si Gd USPIO 118.20 0.00 0.00 2.11 0 0 USPIO-amine 112.78 3.83 0.00 2.019 0.13 0  4% Gd USPIO 114.40 0.00 0.38 2.048 0 0.002  4% Gd USPIO-amine 147.20 3.14 0.57 2.63 0.11 0.0036  6% Gd USPIO 109.54 0.00 0.41 1.96 0 0.0026  6% Gd USPIO-amine 102.70 2.68 5.85 1.83 0.095 0.037  8% Gd USPIO 101.50 0.00 9.20 1.81 0 0.058  8% Gd USPIO-amine 113.14 3.06 9.38 2.02 0.10 0.059 10% Gd USPIO 87.50 0.00 10.53 1.56 0 0.066 10% Gd USPIO-amine 90.72 2.92 7.72 1.62 0.10 0.049

EXAMPLE 3 USPIO-PEG₄₀₀-b-Peptide-mPEG₂₀₀₀ Synthesis of Homobifunctional COOH-PEG₄₀₀-COOH Diacid

The homobifunctional PEG₄₀₀ diacid is synthesized according to the following procedure. Briefly, 20 g of PEG₄₀₀ is dissolved in 300 mL acetone with vigorous stirring in an ice bath. After 15 minutes, 40 mL of Jones reagent (chromium oxide, sulfuric acid, and water) is slowly added and the reaction is stirred for 16 hours. After this time, 20 mL of isopropanol is added to quench the reaction and 2 g of activated carbon is added and stirred for 2 hours. After this time, the reaction mixture is filtered and the solvent is evaporated. The product is obtained from solvent extraction with methylene chloride (CH₂Cl₂) and characterized with ¹H NMR. FIG. 6A shows that PEG methylene (—CH₂) protons, and end methylene (—O—CH₂) protons of PEG₄₀₀ are observed at about 1.35 ppm (a), 4.23 ppm (b) and 3.79 ppm (c) with no impurities observed from the spectrum as expected.

Synthesis of Heterobifunctional HOOC-PEG₄₀₀-NHS

The homobifunctional PEG₄₀₀ diacid synthesized from the previous step (5 g, 8.96 mmol) is reacted with DCC (3.69 g, 17.8 mmol) and NHS (0.896 g, 8.96 mmol) in 100 mL of anhydrous methylene chloride (CH₂Cl₂) and the reaction is stirred for 24 hours. The reaction is quenched with ultra-pure H₂O, filtered, and concentrated. The unreacted NHS is removed by solvent extraction with benzene and the polymers are precipitated in ether. The product is then column chromatographed to obtain pure heterobifunctional COOH-PEG₄₀₀-NHS. The quantitative analysis of the NHS substitution is determined with ¹H NMR, MALDI-TOF and FT-IR.

Synthesis of Blocked Peptides (b-Peptides)

Each of the bioresponsive peptides—ET-1, the RGDS, and the HIV tat peptide are synthesized in tandem with the MMP-13 cleavable sequence, PQGLA, to produce three separate contiguous peptides: GCSCSSLMDKECVYFCHLDIIWGPQGLAG, GRGDSGPQGLAG, and GYGRKKRRQRRRGPQGLAG respectively, in which glycines (gly, G) is added as spacer amino acids to increase enzyme recognition of the peptides. These peptides are synthesized using standard fluorenylmethoxycarbonyl (Fmoc) chemistry on an Applied Biosystems 431A peptide synthesizer (Foster, Calif.). As controls, scrambled peptides are also synthesized and conjugated to nanoparticles in order to demonstrate specificity of the peptides for enzyme cleavage as well as receptor binding and cell penetration.

All peptides are synthesized with an ethylene diamine resin on the C-terminus of the peptides (synthesis occurs from the C— to N-terminus), which after cleaving the peptide from the resin introduces an amine group for conjugation to the carboxylate group of the PEG₄₀₀ linker. Moreover, all peptides are synthesized with ‘blocked’ amino acids in which all reactive side chains of the amino acids are protected. Selective blocking (different blocking groups) and deprotection of the N-terminal glycine facilitates conjugation of the peptide to the outer stealth PEG layer via the N-terminal amine group. After synthesis, the resin of each of the b-peptide is cleaved. Briefly, 800 mg of each peptide is dissolved in 25 mL of 3% trifluoacetic acid in CH₂Cl₂ and the reaction mixture is stirred for 4 hours. After this time, the reaction mixture is filtered to remove the cleaved resin. The cleaved peptides are purified with reverse phase high pressure liquid chromatography as shown in FIGS. 6B-6C for GRGDSGPQGLAG (RGDS) containing peptide.

Synthesis of Ultra Small Paramagnetic Iron Oxide-PEG₄₀₀

Each of the synthesized bioresponsive peptides GCSCSSLMDKECVYFCHLDIIWGPQGLAG, GRGDSGPQGLAG, and GYGRKKRRQRRRGPQGLAG are conjugated to ultra small paramagnetic iron oxide nanoparticles separately via a short (PEG₄₀₀) linker. Briefly, 1 g of amine-functionalized ultra small paramagnetic iron oxide nanoparticles are dispersed in basic water and stirred for 30 minutes under argon gas. After this time, a 3.0 mole excess of COOH-PEG₄₀₀—NHS is added slowly through an addition funnel and stirred overnight. The reaction mixture is acidified, centrifuged to remove unreacted PEG₄₀₀, and washed with ultra-pure water as previously described. The product is characterized for the absence of the amine groups and the presence of PEG₄₀₀ with a ninhydrin assay and FT-IR spectroscopy. These pegylated nanoparticles are then conjugated to the bioresponsive peptides.

Synthesis of Ultra Small Paramagnetic Iron Oxide-PEG₄₀₀-b-Peptide

Briefly, a 3.0 mole excess of RGDS, ET-1, and HIV Tat with MMP-13 cleavable sequence or scrambled peptide sequence of each of the contiguous peptides mentioned above are weighed separately and each are dissolved in 25 mL of CH₂Cl₂ and transferred to separate three-neck round bottom flasks purged with argon gas. After about 30 minutes, 0.5 g of the ultra small paramagnetic iron oxide-PEG₄₀₀ nanoparticles are added to each of the reaction solutions, which are then stirred overnight. The reaction mixtures are then washed separately with water and CH₂Cl₂ to remove unreacted peptide and each of the different peptide-conjugated nanoparticles are washed with 0.1M HCl to quench the reaction, followed with 0.1M NaOH for neutralization. The CH₂Cl₂ is dried with anhydrous MgSO₄, filtered, and the solvent is evaporated. The different peptide-conjugated nanoparticles are then characterized with a standard bicinchoninic assay (BCA) as per the manufacturer's instruction to determine the amount of peptides conjugated per nanoparticle.

Synthesis of Stealth Layer: Heterobifunctional COOH-PEG2000-OMe

Commercially available monomethoxy-PEG (HO-PEG₂₀₀₀-OMe) (MW 2000) is converted to COOH-PEG₂₀₀₀-OMe and conjugated to the bioresponsive peptides (from the N-terminus selectively deprotected glycine) to form an inert outer stealth layer that is released upon subsequent cleavage by MMP-13 enzymes in the tumor to reveal a targeting ligand.

The synthesis of COOH-PEG₂₀₀₀-OMe (outer stealth layer) is performed according to the following procedure. Commercially available monomethoxy-PEG (HO-PEG₂₀₀₀-OMe) (MW 2000) is converted to COOH-PEG₂₀₀₀-OMe and conjugated to the bioresponsive peptides (via N-terminus selectively deprotected glycine) to form an outer stealth layer that is released upon cleavage by MMP-13 enzymes in the tumor to reveal a targeting ligand. This polymer is synthesized and characterized exactly as described above. As can be seen from FIG. 6D, the carboxyl (—COOH), PEG methylene (—CH₂) protons, and end methylene (—O—CH₂) protons of PEG₂₀₀₀ are observed at 4.0 ppm, 3.6 ppm, and 3.3 ppm respectively, with no impurities observed from the spectrum as expected.

Synthesis of Ultra Small Paramagnetic Iron Oxide-PEG₄₀₀-b-Peptide-mPEG₂₀₀₀

Briefly, 0.5 g of each different ultra small paramagnetic iron oxide-PEG₄₀₀-b-Peptide nanoparticles are dissolved in 30 mL CH₂Cl₂ in separate three-neck round bottom flasks purged with argon gas. After 30 minutes, 25% piperidine in dimethylformamide is added to each reaction mixture that is stirred for 2 hours. After this time, a 3 mole excess of COOH-PEG₂₀₀₀-OMe is added to each of the reaction mixture, which is stirred for 4 to 6 hours. The solvent is then evaporated and the products are washed separately to remove unreacted mPEG₂₀₀₀. The final biomimetic ultra small paramagnetic iron oxide-PEG₄₀₀-Peptide-mPEG₅₀₀₀ nanoparticles are obtained by deprotection of the conjugated peptides. Briefly, 0.5 g of each different ultra small paramagnetic iron oxide-PEG₄₀₀-b-Peptide-mPEG₂₀₀₀ nanoparticles are dissolved in 30 ml 50% TFA in CH₂Cl₂ in separate three-neck round bottom flasks under argon gas for 3 to 4 hours. After the reaction time, the nanoparticles are filtered from the solvent containing the protecting groups, and the nanoparticles are washed several times with CH₂Cl₂. The final nanoparticles are then dried in vacuo. These nanoparticles are characterized with TEM and dynamic light scattering to determine the particle size and hydrodynamic volume respectively.

Quantitative Determination of the T2 Relaxivities

MRI experiment are performed on a 3.0 T horizontal bore General Electric Excite HD imaging system, using the standard 8 channel head coil. Multi-Echo (n=8) Spin-Echo images are obtained for each sample set. The TE values for the echo train are centered around the T2 values determined with a large TR=5000 s to exclude any influence of T1 relaxation effects. The T2 relaxation times are calculated from a linear fit of the logarithmic region-of-interest signal amplitudes versus TE using the Matlab (Mathworks Inc.) software. Relaxivity values aree derived according to equation (1)

$\begin{matrix} {\frac{1}{T_{2}} = {\frac{1}{T_{20}} + {r \cdot c}}} & (1) \end{matrix}$

with T₂₀ as the T2 value for the agarose gel, r as relaxivity and c as concentration. Statistical analysis (ANOVA and Student's t test) are performed on each data set.

Agarose phantom gels with and without the nanoparticles are prepared and analyzed with an MRI scanner. Agarose gels are used in these studies to simulate the protons present in a tissue environment. The controls for the experiment include agarose gels only and commercially purchased Ferumoxsil® (Mallinckrodt, Inc., Hazelwood, Mo.) with a crystal size of 10 nm.

A set of seven phantoms are built for these studies that contain agarose gels only, agarose gels with Ferumoxsil®, and agarose gels with varying concentrations of the following: synthesized ultra small paramagnetic iron oxide nanoparticles, amine-functionalized ultra small paramagnetic iron oxide nanoparticles, ultra small paramagnetic iron oxide-PEG₄₀₀ nanoparticles, and each of the different ultra small paramagnetic iron oxide-PEG₄₀₀-Peptide nanoparticles, and ultra small paramagnetic iron oxide-PEG₄₀₀-Peptide-PEG₂₀₀₀ nanoparticles.

The T₂ values for each of the intermediate ultra small paramagnetic iron oxide nanoparticlesm e.g. ultra small paramagnetic iron oxide-PEG₄₀₀, are determined in order to fully characterize the relaxation of the nanoparticles at each modification step. Briefly, Ferumoxsil® and iron oxide nanoparticles at concentrations of 0.001, 0.01, 0.1, 1.0, 10.0, 25.0, 50.0, 75.0, and 100 μg/mL are added to a boiled agarose solution to form a final concentration of 1% agarose gel. The solution is mixed and quickly poured into sterile 15 mL polypropylene tubes that are slowly cooled to room temperature so as to avoid trapping air bubbles inside the tubes. The tubes are then placed in a polystyrene rack that and are secured in a water bath fitted to a MRI head coil.

FIG. 6E shows relaxivities values of control ultra small paramagnetic iron oxide nanoparticles, synthesized ultra small paramagnetic iron oxide nanoparticles (130.8 mmol/s), and amine-functionalized ultra small paramagnetic iron oxide nanoparticles (5.5 mmol/s).

The enzymatic degradation of the nanoparticles with MMP-13 (collagenase-3)-labile domains is assessed by monitoring the kinetics of mass loss of the ultra small paramagnetic iron oxide nanoparticles as a function of time and enzyme concentration. The cleavage of the MMP-13 sequence within the nanoparticle results in the release of the outer stealth PEG layer that corresponds to a molecular weight of about 2000 but produces nanoparticles with a smaller hydrodynamic volume. The specificity of the sequence for cleavage via MMP-13 is investigated with the non-specific enzyme elastase.

Release Kinetics of Outer Peg Layer: MMP-13-Dependent Degradation of Biomimetic USPIO Nanoparticles

The three different biomimetic ultra small paramagnetic iron oxide nanoparticles (ET-1, RGDS, HIV Tat) and their corresponding ultra small paramagnetic iron oxide nanoparticles with scrambled peptide sequences are weighed separately and incubated with varying concentrations of MMP-13 or with elastase. Briefly, 100 mg of each iron oxide nanoparticles are added to separate vials each with 1.0 mL of phosphate buffered saline (PBS) with 0.2 mg/mL of sodium azide and 1 mM calcium chloride (CaCl₂). Four sets of vials are labeled as 0, 8, and 24 hours and 0.025, 0.0050, and 0.0025 mg/mL (collagenase) and 0.025 mg/mL elastase. Crude collagenase or elastase is then added to the vials to produce the enzyme concentrations listed above. Control samples also include untreated biomimetic ultra small paramagnetic iron oxide nanoparticles. All samples are incubated at 37° C. and at predetermined time intervals stated above samples are removed and analyzed with HPLC, MALDI-TOF, and TEM to determine the molecular weight of the released PEG and the nanoparticle size for both treated and non-treated samples. Statistical analysis (ANOVA and Student's t test) is performed on each data set (N=3). Only nanoparticles containing the correct MMP-13 cleavable sequence are cleaved by the enzymes to release the outer PEG₂₀₀₀ layer.

Receptor-Mediated Uptake of Biomimetic USPIO Nanoparticles with ET-1 and RGDS

Cells (OVCA and HUVECs) are seeded at a density of 9×10⁵ cells/well in a 6-well plate and incubated at 37° C. for at least 24 hours prior to treatment with nanoparticles. The cells are untreated or treated with plain, bioresponsive, or scrambled nanoparticles at a concentration of 0.03 μmol/mL. In addition, the cells are simultaneously treated with crude collagenase to facilitate the release of the PEG stealth layer. Controls also include cells not treated with the enzyme. After incubation of the cells at predetermined time intervals of 0 hour, 4 hours, 8 hours, and 24 hours, the cells are washed thrice with 1×PBS (0.1 mol/L, pH 7.4) buffer, trypsinized, centrifuged for 5 minutes at 1500 rpm, and counted with a hemocytometer. A total of 7×10⁵ cells are then mixed with 1% agarose at 37° C. and the T₂ relaxation values are measured with MRI. For the competition assay, either free ET-1 or RGDS ligand at a 10,000:1 ratio (ligand:nanoparticles) is used to pretreat the cells prior to the addition of the nanoparticles and T₂ values are determined.

Receptor-Independent Uptake of Biomimetic Ultra Small Paramagnetic Nanoparticles with HIV Tat

Cells (OVCA and HUVECs) are seeded, treated, and analyzed as described above except that the incubation times are performed at 4° C. and 37° C., to demonstrate that uptake of the biomimetic nanoparticles occurs in a receptor-independent manner in which receptor-mediated uptake is inhibited at 4° C.

For Prussian blue staining, cells are plated and treated with plain, bioresponsive, or scrambled nanoparticles as described above except that glass coverslips is placed in the 6-well plates prior to seeding the cells and the cells are incubated for 24 hours. After incubation, the coverslips are washed with PBS and fixed with the organic solvents methanol and acetone. Briefly, the coverslips are added to cooled (−20° C.) in methanol for 10 minutes. The coverslips are then removed from the methanol and the cells are permeabilized with cooled acetone (−20° C.) for 1 minute. The coverslips are then added to 10% potassium ferrocyanide for 5 min and 10% potassium ferrocyanide in 20% hydrochloric acid for 30 minutes, and the nuclei are counterstained by adding the coverslip to a solution of propidium iodide at a concentration of 0.01 mg/mL. The coverslips are then washed thrice with 1×PBS buffer to remove excess propidium iodide and analyzed with microscopy to show uptake of the bioresponsive nanoparticles that are apparent as blue granules.

Cytotoxicity of Biomimetic Ultra Small Paramagnetic Iron Oxide Nanoparticles

Cells (OVCA and HUVECs) are seeded and treated as described above. After the incubation times, the cells are treated with MTT with the method of Mossman. Briefly, the cells are washed with 1×PBS and then incubated with a solution of MTT in 1×PBS buffer at a concentration of 2 mg/mL. The plates are then incubated at 37° C. for 4 hours, after which the medium is removed and 1.0 mL of dimethylsulfoxide is added to the wells to dissolve the formazan crystals. The absorbance of the samples is determined at 570 nm and the readings are normalized to that of the untreated cells.

EXAMPLE 4 USPIO-TEG-Peptide-PEG Contrast Agent

The synthesis of biomimetic USPIO-TEG-Peptide-mPEG₅₀₀₀ is depicted in FIGS. 7A-7E.

Synthesis of HO-TEG-Ots

10 gms (0.0255 moles) of TEG was weighed in a 3 neck RBF purged with nitrogen. To the mixture 4.86 gms of Tosylchloride was added at 0° C. and the mixture was stirred for 3 hours. At the end of three hours 0.85 mls of TEA was added to neutralize the acid formed and the reaction mixture was left to stir overnight. The progress of the reaction was then monitored by TLC. After the completion of the reaction the solvent was evaporated and the sample was dried under vacuum. The crude product was then chromatographed using ethyl acetate as the solvent. Initial fractions were then left out and the fractions containing pure product were then pooled together and solvent was evaporated and kept in vacuum. Sample obtained was 3.5 gms and NMR showed pure product as shown in FIG. 8A.

Synthesis of HO-TEG-N₃

1.5 gms of HO-TEG-Ots was dissolved in 25-30 mls of DMF followed by the addition of 0.007 gms of sodium azide. The reaction mixture was refluxed and progress of the reaction was monitored by TLC. The TLC showed the formation of the product and the completion of the reaction using 2% methanol in methylene chloride NMR in FIG. 8B shows the presence of the pure product.

Synthesis of N₃-TEG-t-butyl Ester

0.047 gms of potassium t-butoxide (M.W: 111.22, 0.425 mmoles) was weighed under nitrogen and transferred to a 50 ml round bottom flask containing 10 mls of anhydrous THF purged with N₂. After 30 mins, 1 gm of N₃O-TEG-OH (0.425 mmoles) was added slowly to the solution using a syringe. Reaction mixture was allowed to react under the nitrogen atmosphere for almost 3-4 hours. After all the solid went into the solution, 0.064 gms t-butyl acrylate (M.W: 128.17, 0.5 mmoles) was added slowly to the solution using a syringe. On the addition of t-butyl acrylate the reaction temperature increased and care was taken to maintain at ambient. The reaction mixture was allowed to stir overnight. The progress of the reaction was monitored by TLC. After the completion of the reaction, the solvent was evaporated and the oily residue along with the solid obtained was dissolved in mixture of ethyl acetate and water (50:50) and layers were separated. The reaction mixture was then purified using column chromatography using Ethyl acetate/Hexane as the solvent. Various fractions which seemed similar in TLC were pooled together and the product obtained was characterized by NMR. NMR indicated the presence of pure product (FIG. 8C).

Synthesis of NH₂-TEG-t-butyl Ester

1.5 gms of N₃-TEG-t-butyl ester was weighed under nitrogen and transferred to a 50 ml round bottom flask containing 25 mls of anhydrous THF purged with N₂. After 30 mins, 1.2 moles of triphenyl phosphine was added slowly to the reaction mixture. Reaction mixture was allowed to react overnight under the nitrogen atmosphere. The progress of the reaction was monitored by TLC (Methanol/Ethyl Acetate 1/10). After the completion of the reaction, the solvent was evaporated and the oily residue was then purified using column chromatography. Various fractions which seemed similar in TLC were pooled together and the product obtained was characterized by NMR. NMR indicated the presence of pure product (FIG. 8D).

Synthesis of HOOC-TEG-t-butyl Ester

FIG. 7A is the synthetic scheme for amine-functionalized iron oxide nanoparticles. FIG. 7B is the synthetic scheme for HOOC-TEG-t-butyl ester. 1.5 gms of NH₂-TEG-t-butyl ester was weighed under nitrogen and transferred to a 50 ml round bottom flask containing 25 mls of anhydrous THF purged with N₂. After 30 mins, 1.2 moles of succinic anhydride was added slowly to the reaction mixture. Reaction mixture was allowed to react overnight under the nitrogen atmosphere. The progress of the reaction was monitored by TLC (Methanol/Ethyl Acetate 1/10). After the completion of the reaction, the solvent was evaporated and the oily residue was then purified using column chromatography. Various fractions which seemed similar in TLC were pooled together and the product obtained was characterized by NMR. NMR indicated the presence of pure product (FIG. 8E).

Purification of Peptide

FIG. 7C is the synthetic scheme for peptide synthesis. Peptide was synthesized using solid phase peptide synthesizer on a trityl chloride resin. The purification of peptide was done by recrystallization and extraction of the peptide in methylene chloride and water. 10 mg of peptide was dissolved in methylene chloride was extracted with water. This extraction was repeated several times followed by the drying of water using methanol. Once water was dried off the solution was then used for MALDI analysis. The MALDI analysis gave the pure product with minute amount of impurities (FIG. 8F).

Synthesis of USPIO-TEG-t-butyl Ester

30 mg of HOOC-TEG-t-butyl ester was mixed with 15.7 mg of EDC in 1 ml of MES buffer and the reaction mixture was heated at 50° C. for about 15-20 mins. This mixture was then added to the suspension of 13 mg of USPIO-amine in 3 mls of water and the reaction mixture was shaken on the shaker for 12 hours. After 12 hours the reaction mixture was centrifuged and washed with water and the solid was separated using magnet and kept in lypholyzer.

Synthesis of USPIO-TEG-Peptide

15 mg of USPIO-TEG t-butyl ester was first hydrolyzed with mixture of methylene chloride and TFA (50:50) for about 2 hours and the solvent was evaporated. After the evaporation of the solvent the sample was kept in lypholyzer for further elemental analysis using EDS. Part of the material (USPIO-TEG)-15 mg was mixed with 15.7 mg of EDC in 1 ml of MES buffer and the reaction mixture was heated at 50° C. for about 15-20 mins. To this mixture 50 mgs of peptide was then added along with acetonitrile to make the peptide more soluble into the reaction solution. The reaction mixture was shaken on the shaker for 12 hours. After 12 hours the reaction mixture was centrifuged and washed with water and the solid was separated using magnet and kept in lypholyzer.

Synthesis of USPIO-TEG-Peptide-mPEG₅₀₀₀

25 mg of USPIO-TEG-peptide was mixed with 23.7 mg of EDC in 1 ml of MES buffer and the reaction mixture was heated at 50° C. for about 15-20 mins. To the reaction mixture 50 mg of PEG-NH₂ was then added along with 1 ml of water. The reaction mixture was shaken on the shaker for 12 hours. After 12 hours the reaction mixture was centrifuged and washed with water and the solid was separated using magnet and kept in lypholyzer. Finally the USPIO-TEG-Peptide-PEG was totally deprotected by stirring the in the solution of 1:1 [TFA:CH₂Cl₂] for 4 hours. The solvent was evaporated and the product obtained was washed with methylene chloride and reprecipitated using a very powerful magnet while the solution was decanted. This procedure was repeated 3 times so as to make sure that all the impurities are being washed out. FIGS. 7D-7E are synthetic schema for the synthesis of USPIO-TEG-Peptide-mPEG₅₀₀₀.

EXAMPLE 5 Dual Functioning Nanoparticles

A dual magnetic resonance imaging (MRI) contrast agent and nonviral gene delivery system comprises iron oxide gadolinium (Gd[III])-doped nanoparticles, coated with an inert layer of gold (Au) are conjugated to a novel biodegradable cationic polymer disulfide-reducible, linear, L-lysine-modified copolymers (LLC). The cationic nanoparticles are transfect mesenchymal stem cells (MSCs) with the IL-12 gene or other anti-tumor gene (FIG. 9).

Synthesis of Functionalized Gold-Coated Gdf[III]-Doped Iron Oxide Nanoparticles

Gd[III]-doped iron oxide (Fe₃O₄) nanoparticles are oxidized to Fe₂O₃ by boiling in nitric acid (HNO₃), and are then coated with gold using a hydroxylamine seedling method with chloroauric acid (HAuCl₄). Six different reaction mixtures ranging from 0 to 10% of Gd[III] ions are prepared. As can be seen from the transmission electron microscopy (TEM) image of the 4% Gd-doped nanoparticles in FIG. 10A, the synthesized particles are spheroidal in shape with a particle size of ˜7-10 nm. In addition, iron oxide nanoparticles with 0, 2, and 4% Gd[III] ions were analyzed with a 3.0 T Horizontal Bore General Electric Excited HD imaging system to determine if the contrast agents could be detected. As can be seen from FIG. 3, all of the newly synthesized nanoparticles could be detected with MRI (copper sulfate (CuSO4) samples were used as phantom controls) (FIG. 10B). Suspended nanoparticles are reacted with 100 mg of SH-PEG-NH₂ with stirring for 12 hr with methods used in our laboratory and purified with a magnet.

Synthesis of Au-Coated Reducible LLC Conjugated Nanoparticles without Ethylenediamine

L-lysine HCl and CBA are added to suspended nanoparticles in (80/20 v/v) of methanol/water (MeOH/H₂O) with stirring in the dark under nitrogen. Conjugated nanoparticles are purified with a magnet. To investigate the feasibility of the reducible LLC polymers binding DNA, linear copolymers not conjugated to the nanoparticles were synthesized. FIG. 11 shows the ¹H NMR of 25% N-boc conjugated polymer with all of the expected peaks of the final product.

Conjugation of N-Boc Ethylenediamine to Au-Coated Reducible LLC Nanoparticles

Reducible LLC conjugated Au-coated nanoparticles suspended in water are mixed with a molar excess of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-boc ethylenediamine with stirring in an oil bath at 40° C. in the dark under nitrogen. Conjugated nanoparticles are purified with a magnet. The acid-liable N-boc amine protection group present on the conjugated ethylenediamine are removed with a (TFA)/H₂O mixture (75/25 v/v) trifluoroacetic acid.

Gel Retardation Assay

To evaluate the ability of the conjugated nanoparticles to complex plasmid DNA, a previously published gel retardation assay is performed with a plasmid coding for the luciferase gene (pCMV-Luc). FIG. 12 shows the synthesized LLC polymers were able to successfully condense plasmid DNA from a 25/1 N/P ratios (nitrogens of polymer/phosphates of DNA).

Reduction of Au-Coated Reducible LLC Nanoparticles

To a solution of conjugated nanoparticles, 1,4-dithio-DL-threitol (DTT) is added and the solution is incubated at room temperature. The reduction of the disulfide bonds in the cationic polymers is monitored with UV spectroscopy to determine the mechanism of reaction.

Determination of T₁ and T₂ Relaxations

The longitudinal (T₁) and transverse (T₂) relaxations of the Au-coated nanoparticles are determined in agarose phantoms. Commercially purchased Ferumoxsil® (Mallinckrodt, Inc., Hazelwood, Mo.) with a crystal size of 10 nm is used as a control.

Transfection Efficiency and Cytotoxicity

Cells are plated and transfected with the contrast agents, i.e., nanoparticles and DNA with luciferase The luciferase assay is performed as previously published. Cells are plated and treated with optimized contrast agents and analyzed with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for mitochondrial damage and a lactate dehydrogenase assay for cytosolic damage as previously published.

MR Detection of Molecular Probes within Cells

To image MR probes within cells, MSCs treated with the optimized contrast agents are fixed and mixed with an agarose phantom to determine whether or not there are differences in the T₁ and T₂ values of the nanoplexes following internalization by MSCs on the MRI scanner.

Transwell Migration Studies

The migration of transfected MSCs to plated 4T1 murine metastatic mammary breast cancer cells is studied using cells plated in a transwell cell culture dish. Cells are analyzed with microscopy.

In Vivo Mouse Model

A metastatic mouse model based on 4T1 cells injected subcutaneously in Balb/c mice is established as previously published. MSCs transfected with optimized contrast agents are injected intravenously via the tail vein after tumors have a volume of 75-120 mm³ and lung metastases have been detected. Animals are anesthetized and imaged as a function of time. After this, animals are treated and imaged twice a week for three weeks, blood is sampled and is analyzed for IL-12 expression with an enzyme-linked immunosorbent assay (ELISA). The animals are then sacrificed and tissue sections are obtained for analysis with hematoxylin and eosin.

The following references were cited herein:

-   1. Young Soo Kang et al. Chem. Mater, 8:2209-2211, 1996. -   2. Cozzoli et al. Chem Soc Rev, 35(11): 195-208, 2006. -   3. Pankhurst et al. J. Phys. D: Appl. Phys. 36:R167, 2003. -   4. West, J. L. and Halas, N. J. CurrOpin Biotechnol, 11(2):215-7,     2000. -   5. Yu et al. IEEE Transactions on Magnetics, 43(6):2436-2438, 2007. -   6. Yabin Sun et al., Chem. Commun., 2006:2765-2767, 2006. -   7. Dodson, J. Gene Ther, 13:283-287, 2006. -   8. Rudge et al. J. Controlled Release, 2001(74):335-340, 2001. -   9. Mah et al., Mol. Ther. 6:106, 2002. -   10. Ito et al. J. Biosci. Bioeng, 1:100, 2005. -   11. Osaka et al. Anal. Bioanal. Chem, 384:593, 2006. -   12. Dobson, J., Gene Ther 13:283, 2006. -   13. Wang Y X, H. S., Krestin G P, Eur Radiol, 11:2319-2331, 2001. -   14. Pilgrimm, H., 2003, USA, 6638494 -   15. LaConte et al. Journal of Magnetic Resonance Imaging,     26:1634-1641, 2007. -   16. Drake et al. Journal of Material Chemistry, 17:4914-4918, 2007. -   17. Hong et al. Chemistry Letters, 33(11):1468-1469, 2004.

Any patents or publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. Further, these patents and publications are incorporated by reference herein to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

One skilled in the art will appreciate readily that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those objects, ends and advantages inherent herein. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art. 

1. A biomimetic contrast agent, comprising: an amine-functionalized iron (II) oxide/iron(III) oxide nanoparticle core; a targeting ligand attached to the nanoparticle core via a linker; an inert outer layer of a hydrophilic polymer conjugated to the targeting ligand.
 2. The biomimetic contrast agent of claim 1, further comprising a metal doping agent in the nanoparticle core.
 3. The biomimetic contrast agent of claim 2, wherein the metal doping agent is gadolinium, manganese, cobalt, or ruthenium.
 4. The biomimetic contrast agent of claim 1, further comprising a gold coating on the nanoparticle core.
 5. The biomimetic contrast agent of claim 1, wherein the ratio of the iron (II) to the iron(III) in the nanoparticle core is about 1:1.
 6. The biomimetic contrast agent of claim 1, wherein the linker comprises a triethylene glycol polymer or a polyethylene glycol polymer.
 7. The biomimetic contrast agent of claim 1, wherein the targeting ligand comprises a contiguous peptide sequence of an enzyme cleavable sequence and a targeting sequence in tandem.
 8. The biomimetic contrast agent of claim 7, further comprising a blocking agent bound to a terminal sidechain amino acid of the peptides.
 9. The biomimetic contrast agent of claim 7, wherein the enzyme cleavable sequence is cleavable by an enzyme associated with ovarian or breast cancer cells.
 10. The biomimetic contrast agent of claim 7, wherein the enzyme cleavable sequence is a metalloproteinase-13 cleavable sequence.
 11. The biomimetic contrast agent of claim 10, wherein the MMP-13 cleavable sequence is shown in SEQ ID NO:
 1. 12. The biomimetic contrast agent of claim 7, wherein the targeting sequence is a endothelin-1 (ET-1) sequence, an integrin-binding sequence or a human immunodeficiency virus Tat peptide sequence.
 13. The biomimetic contrast agent of claim 12, wherein the ET-1 sequence is shown in SEQ ID NO: 2, the integrin-binding sequence is shown in SEQ ID NO: 3 and the HIV Tat sequence is shown in SEQ ID NO:
 4. 14. The biomimetic contrast agent of claim 7, wherein the targeting ligand sequence is shown in SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO:
 7. 15. The biomimetic contrast agent of claim 1, wherein the inert outer layer comprises a polyethylene glycol polymer.
 16. A kit comprising the biomimetic contrast agent of claim
 1. 17. An in vivo method using magnetic resonance imaging for detecting an early stage primary or metastatic cancer in a subject, comprising: administering to the subject a sufficient amount of the biomimetic contrast agent of claim 1 to provide a detectable contrast image of the contrast agent within the primary or metastatic cancer therein, wherein a location of the image correlates to a location of the cancer.
 18. The method of claim 17, wherein the primary or metastatic cancer is ovarian cancer or breast cancer.
 19. A contrast agent, comprising: a metal-doped iron (II) oxide/iron(III) oxide nanoparticle core; an inert layer of gold coating the nanoparticle core; and a biodegradable cationic polymer linked thereto.
 20. The contrast agent of claim 19, further comprising DNA encoding an anti-tumor cytokine complexed with the cationic polymer.
 21. The contrast agent of claim 20, wherein the anti-tumor cytokine is interleukin-12.
 22. The contrast agent of claim 19, wherein the doping metal is gadolinium (III), manganese, cobalt, or ruthenium.
 23. The contrast agent of claim 19, wherein the cationic polymer comprises disulfide-reducible, linear, L-lysine-modified copolymers (LLC).
 24. A kit comprising one or both of the dual contrast agent of claim 19 or the dual contrast agent complexed to a DNA encoding an anti-tumor cytokine.
 25. The kit of claim 24, further comprising the buffers and reagents effective to transfect mesenchymal stem cells with the contrast agent-DNA complex.
 26. A dual contrast agent and gene delivery system, comprising: mesenchymal stem cells (MSCs) transfected with the contrast agent of claim 19 complexed with a DNA encoding an anti-tumor cytokine.
 27. The system of claim 26, wherein the anti-tumor cytokine is interleukin-12.
 28. An method for reducing metastasis of tumor cells in a subject, comprising: administering to the subject an amount of the mesenchymal stem cells (MSCs) comprising the dual contrast agent and gene delivery system of claim 26 sufficient to target metastatic tumor cells; simultaneously delivering the contrast agent and the anti-tumor cytokine thereto; and obtaining a magnetic resonance image of the contrast agent within the metastatic tumor cells; wherein simultaneously imaging the contrast agent and delivering the anti-tumor cytokine locates a site of metastatic tumor cells and induces a pro-inflammatory response against the same, thereby reducing metastasis of the tumor cells.
 29. The method of claim 28, further comprising: implementing one or both of a surgical regimen or one or more other chemotherapeutic regimens. 